A method for inhibiting the expression of a target gene in cells, tissues, or individuals includes an approach by which double-stranded RNA is introduced into the cells, tissues, or individuals. By this introduction of double-stranded RNA, mRNA having homology to the sequence is degraded such that the expression of the target gene is inhibited. This effect is called “RNA interference” or “RNAi”. RNA interference was originally reported in C. elegans (see e.g., Non Patent Reference 1) and then also reported in plants (see e.g., Non Patent Reference 2).
Double-stranded RNA consisting of 21-nucleotide sense and antisense strands having a 2-nucleotide overhang at the 3′-end (small interfering RNA: siRNA) has been reported to have an RNA interference effect in cultured cells of vertebrates (see e.g., Non Patent Reference 3).
siRNA is useful for the identification of gene functions, screening of cell strains suitable for useful substance production, regulation of genes involved in disease, etc., but, however, it is disadvantageously degraded easily by RNase (see e.g., Non Patent Reference 4). RNA synthesis is more difficult than DNA synthesis and therefore reportedly entails a 5 to 10-times higher cost (see e.g., Non Patent Reference 5). Exemplary reasons for this include the need for a protective group for the 2′-hydroxy group, the need to deprotect the protective group, and poor RNA synthesis yields attributed to a reduction in condensation yield due to the steric hindrance of the protective group (see e.g., Non Patent Reference 6).
Thus, there has been demand for a polynucleotide to be developed which is highly resistant to RNase, can be produced at low cost, and retains RNAi activity.
To obtain siRNA having resistance to RNase, a method has been researched in which all or some of the RNA nucleotides constituting an siRNA are substituted with 2′-deoxyribonucleotides (DNAs) (see e.g., Patent Reference 1 and Non Patent References 7, 8, 9, 10, and 11). However, siRNA having both resistance to RNase and an RNA interference effect equivalent to that of natural siRNA has not been obtained yet.
An oligonucleotide having a phosphorothioate (PS) bond in which the non-bridged oxygen atom of the phosphate group in the phosphodiester bond has been substituted with a sulfur atom is known to be resistant to nuclease (see e.g., Non Patent Reference 12). siRNA having PS bonds substituting the phosphodiester bonds has been reported to exhibit RNA interference equivalent to that of unmodified siRNA (see e.g., Non Patent References 9, 13, and 14). However, the increased number of PS bonds in an oligonucleotide causes thermodynamic instability of double-stranded RNA and nonspecific binding with proteins and is therefore not thought to be preferable (see e.g., Non Patent Reference 15).
An attempt has also been made to obtain stable siRNA by substituting natural RNA with modified RNA. Since the 2′-OH group of RNA is essential for the RNase degradation reaction, this 2′-OH group is alkylated such that it does not serve as a substrate for RNase. A large number of such 2′-O-alkyl nucleoside derivatives have been reported. 2′-O-methyl nucleotides are naturally occurring modified nucleotides also found in tRNA and have been studied since the early stages of antisense research (see e.g., Non Patent Reference 16).
It has been reported that RNAi is completely lost by substituting either or both of the sense and antisense strands of siRNA with 2′-O-methyl nucleotides (see e.g., Non Patent References 7, 17, and 18) or that only weak RNAi is observed when substituting all ribonucleotides in the sense or antisense strand of siRNA with 2′-O-methyl nucleotides, and that RNAi is completely lost by substituting both the strands therewith (see e.g., Non Patent Reference 9).
There is a report that when all RNAs in a sense strand are substituted with 2′-O-methyl nucleotides, RNAi equivalent to that of unmodified siRNA is obtained, but this is influenced by the sequence of siRNA used in the experiment (see e.g., Non Patent Reference 19).
It has been reported that when four 2′-O-methyl nucleotides are introduced to the end of an siRNA, its RNAi is retained (see e.g., Non Patent Reference 14) and that when 2′-O-methyl nucleotides are alternately introduced to both the ends of the sense and antisense strands of siRNA, RNAi equivalent to that of unmodified siRNA is obtained (see e.g., Non Patent Reference 18). Moreover, it has been reported that the introduction of 3 consecutive 2′-O-methyl nucleotides does not cause any reduction in activity for a sense strand but does cause a reduction in activity for an antisense strand, and in particular, its introduction to the 5′-end of the antisense strand significantly reduces activity (see e.g., Non Patent Reference 20).
Moreover, siRNA comprising 2′-deoxyribonucleotides in the vicinity of the 3′ and 5′-ends of the sense strand and 2′-O-methyl nucleotides in the central portion thereof has been reported but has not been compared in RNAi activity with unmodified siRNA (see e.g., Non Patent Reference 21).
An oligonucleotide having an artificially synthesized modified RNA 2′-deoxy-2′-fluoronucleotide (2′-F) preferentially forms the same N-type conformation as that of ribonucleotides and has higher affinity for RNA (see e.g., Non Patent Reference 22). However, those having phosphodiester bonds have no resistance to nuclease and therefore, in order to have nuclease resistance, they must be substituted with phosphorothioate bonds therefor (see e.g., Non Patent Reference 22).
It has been reported that when a pyrimidine nucleotide in siRNA is substituted with 2′-F, the resulting siRNA exhibits RNAi equivalent to that of unmodified siRNA (see e.g., Non Patent References 9 and 14). The introduction of 3 consecutive 2′-F moieties to an antisense strand hardly reduces its activity (see e.g., Non Patent Reference 20). Moreover, it has been reported that when either a pyrimidine nucleotide or a purine nucleotide in sense and/or antisense strands is substituted by 2′-F and both the modified strands are combined, the resulting siRNA exhibits RNAi equivalent to that of unmodified siRNA (see e.g., Non Patent Reference 23).
However, among these, those exhibiting an RNA interference effect contain a ribonucleotide and are thus degraded by RNase. It has been reported that when a pyrimidine nucleotide in siRNA was substituted by 2′-F, the enhancement of RNAi or its prolonged effect was not observed in animal models (see e.g., Non Patent Reference 24). Moreover, it has been reported that nonnatural nucleosides 2′-deoxy-2′-fluorocytidine and 2′-deoxy-2′-fluorouridine, which allegedly exhibit no toxicity when administered to rats or woodchucks, serve as substrates for DNA polymerase or RNA polymerase through intracellular triphosphorylation and are incorporated into DNA, RNA, and mitochondrial DNA in various organs (see e.g., Non Patent References 25 and 26). A triphosphate form of 2′-deoxy-2′-fluoronucleoside is incorporated as a substrate for DNA polymerase α or γ into DNA, whereas a triphosphate form of 2′-O-methyl nucleoside has been confirmed in vitro not to serve as a substrate for DNA polymerase α or γ (see e.g., Non Patent Reference 27). The genetic toxicity of the 2′-deoxy-2′-fluoronucleoside is of concern (see e.g., Non Patent Reference 28).
It has been reported that when all nucleotides in siRNA are substituted with 2′-F, its RNAi is merely slightly lower than that of unmodified siRNA and that such siRNA is resistant to RNase (see e.g., Non Patent Reference 29).
It has been reported that when 2′-O-methyl nucleotides and 2′-F are alternately introduced into the sense and antisense strands of siRNA, the obtained siRNA has RNAi equivalent to or higher than that of unmodified siRNA and is relatively stably maintained in serum (see e.g., Non Patent Reference 30). However, cytotoxicity or side effects caused by the introduction of a large number of normatural nucleic acids is of concern.
ENAs (2′-O,4′-C-ethylene-bridged nucleic acids) are modified nucleic acids having stability to nuclease (see e.g., Non Patent References 31 and 32). It has been reported that when ENAs are introduced to replace 2 nucleotides in the 3′-terminal overhang site of either or both of the sense and antisense strands of siRNA, the RNAi activity is reduced (see e.g., Non Patent Reference 33).
It has been reported that the introduction of chemically synthesized siRNA into cells phosphorylates the 5′-ends of both sense and antisense strands (see e.g., Non Patent Reference 34). In human cells, RNA kinase hClp1 has been reported to be responsible for the 5′-phosphorylation of siRNA (see e.g., Non Patent Reference 35). When siRNA having a phosphorylated 5′-end and siRNA having an unphosphorylated 5′-end were separately introduced into cells and their RNAi activity compared, no difference in activity was seen therebetween, indicating that siRNA having an unphosphorylated 5′-end is easily subject to phosphorylation in cells (see e.g., Non Patent Reference 14).
The X-ray analysis of a complex of an antisense strand with Argonaute protein (Ago) known to participate in RNAi activity has showed that the 5′-terminal phosphate group of the antisense strand and its neighboring nucleotides are strongly bound by the PIWI domain of Ago (see e.g., Non Patent Reference 36).
As regards the chain length of siRNA, 21 nucleotides are routinely used with each of the sense and antisense strands having a 2-nucleotide overhang at the 3′-end. When an antisense strand is set to being 21 nucleotides in length and the chain length of a sense strand is varied from the 3′ or 5′-end, siRNA having a 21-nucleotides sense strand has been shown to have the strongest RNAi activity (see e.g., Non Patent References 7 and 37). Moreover, it has been reported that when a sense strand is 3′-terminally truncated to 17 or 18 nucleotides in chain length, the resulting siRNA exhibits RNAi activity equivalent to that of siRNA having a 21-nucleotide sense strand (see e.g., Non Patent Reference 38).
siRNA consisting of 21 nucleotides has been shown to have the strongest RNAi activity when the length of the 3′-terminal overhang is 2 nucleotides (see e.g., Non Patent Reference 7). It has been reported that when RNAi activity was examined using siRNA having the sequence AA, CC, GG, UU, or UG (wild-type) or TdG or TT (T and dG are 2′-deoxyribonucleotides) as the 3′-terminal overhang, all the sequences had RNAi activity (see e.g., Non Patent Reference 7). Moreover, it has been reported that siRNA having a UU sequence as the 3′-terminal overhang exhibits higher RNAi activity than that of siRNA having a TT sequence (see e.g., Non Patent Reference 10).
Double-stranded RNA such as polyl:polyC has been known as an interferon inducer for a long time, and TLR3 (Toll-like receptor 3) is involved in the mechanism. siRNA is also known to be recognized by TLR3 and its family members TLR7 and TLR8 are known to induce interferon or cytokines. Particularly, siRNA having a GU, UGUGU, or GUCCUUCAA sequence has been reported to tend to cause an immune response (see e.g., Non Patent References 39, 40, and 41). Moreover, the introduction of DNAs or chemically modified nucleotides such as 2′-OMeRNAs into siRNA has been shown to inhibit such immune response (see e.g., Non Patent References 41, 42, and 43).
The present inventors have conducted diligent studies to obtain a polynucleotide that is resistant to RNase, can be synthesized at low cost, and has an RNA interference effect, and have consequently completed the present invention by finding that a double-stranded polynucleotide comprising an oligonucleotide unit of DNAs and 2′-O-methyl RNAs alternately combined can solve the problems described above.